CN105986001B - A kind of high-throughput prey antagonist screening technique based on embrane-associated protein and fluorescence complementary - Google Patents
A kind of high-throughput prey antagonist screening technique based on embrane-associated protein and fluorescence complementary Download PDFInfo
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Abstract
A kind of high-throughput prey antagonist screening technique based on embrane-associated protein and fluorescence complementary.The present invention relates to genetic engineering fields, specifically disclose a kind of new method and its application for screening receptor antagonist.The method will be present in extracellular ligand and receptor and reach cell membrane by signal peptide amalgamation and expression Policy Table, and to be retained in the ligand domain that receptor interacts on the outside of cell membrane, the ligand is coupled by transmembrane peptides and effect protein intracellular, the generation or antagonism that ligand and receptor interact on the outside of film are coupled at intracellular effect protein and transform into macrobiology effect, to be the screening providing method of those secreting type ligands and receptor antagonist.
Description
Technical field
The present invention relates to genetic engineering fields, specifically disclose the new method and its application of screening receptor antagonist.
Background technique
Antagonist (antagonist) refers to can be in conjunction with receptor, but does not have a kind of object of activated receptor biological function
Matter, biological function and agonist (agonist) inhibit downstream physiological reaction, are one on the contrary, by the transduction of disabling signal
A important medicament categories.Antagonist is from broadly including capableing of small molecule compound, the recombination that antagonistic protein interacts
Protein, antibody etc., wherein small molecule compound antagonist is most widely used.The antagonist screening of receptor has weight in medicament research and development
Want meaning.Such as antagonist AMD3100, ALX40-4C of CXCR4, it can be interacted and be inhibited by antagonism CXCL12 and CXCR4
The tumour growth of part CXCR4 related neoplasms；Endothelial growth factor receptor EGFR antagonist Gefitinib (trade name) is for leading to
It crosses inhibition of angiogenesis and inhibits tumour.
At present for the High Throughput Screening Assay of receptor antagonist include two kinds of strategies: the first, detection compound to by
The strategy of body downstream biochemical reactions.This method needs using different detection methods, example the receptor of different function
Such as, for studying calcium channel receptor antagonist, using calcium ion fluorescent detection system；In another example for research
GPCR receptor antagonist, using cAMP detection system etc., so applying without universality, and due to receptor and downstream
The cross reaction of signal path, so that there are problems for the detection specificity of this detection system.Second, directly detection ligand-
The strategy that receptor combines.It is this strategy by the methods of radioactive label, enzyme-linked tag, membrane flexibility directly research ligand and
The interaction of receptor.Such methods need to purify active ligand albumen, and the operation such as need that ligandin is marked,
Activity is relatively difficult to guarantee, and repeatability is not strong.
Screening technique for soluble recepter antagonist intracellular it is proposed that the method based on traditional yeast double cross,
But the receptor and ligand that traditional yeast two-hybrid method is studied are present in cell interior, and compound allows for being efficiently entering thin
The blocking effect intracellular that can have an opportunity, but in high flux screening, the factor is difficult to control, therefore can not be sufficiently sharp
With entire compound library.
It can be seen that a kind of easy, pervasive, high-throughput receptor antagonist detection method is urgently developed.
Summary of the invention
It is an object of the invention to overcome the deficiencies of existing technologies, provide it is a kind of for be present in extracellular ligand screening by
The method of body antagonist, the method will be present in extracellular ligand and receptor by the outer yeast two-hybrid system of film express to
Cell membrane, and to be retained in the ligand that receptor interacts on the outside of cell membrane, the ligand passes through transmembrane peptides and born of the same parents
Internal effect albumen coupling, thus short of money for the ligand screening receptor of those N, C-terminal simultaneously on the outside of cytoplasma membrane or in endoplasmic
Anti-agent.As shown in Figure 1, ligand and receptor both sides interact in the presence of no antagonist, effect protein knot intracellular is drawn
Structure domain is complementary, and recombination becomes effect protein, generates Physiological and Biochemical effect.When antagonist is added, ANTAGONIST COMPETITIVE with by
Body combines, the complementary effect of antagonism effect protein intracellular.With the increase of Antagonist concentration, complementary effect protein total amount occurs
It decreases, is in inverse S-curve to Antagonist concentration mapping, can get the median effective dose of antagonist by formula fitting
IC50。
The present invention is achieved by the following technical solutions:
The first aspect of the present invention discloses a kind of method to be present in extracellular ligand screening receptor antagonist, institute
The method of stating specifically comprises the following steps:
(1) it using ligand as bait, constructs bait expression vector: secreting signal peptide coding gene sequence, transmembrane peptides is compiled
At least one of code gene order is melted with bait coding gene sequence and effect protein structural domain coding gene sequence intracellular
Bait expression unit sequence is obtained after conjunction, and the bait expression unit is cloned into the expression plasmid of yeast with screening label
On, building obtains the bait expression vector of expression bait；
(2) it using receptor as prey, constructs prey expression vector: secreting signal peptide coding gene sequence, transmembrane peptides is compiled
At least one of code gene order is melted with prey coding gene sequence and effect protein structural domain coding gene sequence intracellular
It closes or the prey expression unit sequence is by prey coding gene sequence and effect protein structural domain encoding gene intracellular
Sequence directly merge after obtain prey expression unit sequence, and by the prey expression unit be cloned into have can screen label
On expression plasmid of yeast, building obtains the prey expression vector of expression prey；
(3) by gained prey expression vector in gained bait expression vector in step (1) and step (2) be transferred in yeast into
Interaction occurs outside cell membrane for row coexpression, expressed ligand and receptor, and it is complementary to draw effect protein intracellular；
(4) antagonist activities are detected as measurement complementary effect: bait table obtained by obtained stable expression step (1)
Up to antagonist candidate is added in the positive yeast that the expression of gained prey carries in carrier and step (2), the half of candidate is calculated
Amount of suppression IC50, thus it is speculated that the affinity Ki or dissociation COEFFICIENT K D of candidate and ligand filter out the receptor antagonist of ligand.
Preferably, the secreting signal peptide refer to one kind can pilot protein matter enter endoplasmic reticulum, and secreted in turn to born of the same parents
Outer amino acid sequence.The secreting signal peptide is the native sequences for being present in mammalian cell, plant cell, yeast cells
Or the sequence that can be completed Protein secretion and go out born of the same parents of engineer.
It is furthermore preferred that the secreting signal peptide is the Wbp1 signal peptide sequence from yeast cells, amino acid sequence is such as
Shown in SEQ ID NO.1, specifically:
MARVMRTDWNFFFCILLQAIFVVGTQTSRTLVLYSK。
Preferably, the transmembrane peptides refer to that a kind of hydrophobicity is extremely strong, can be fitted into fusion protein to the ammonia of cytoplasma membrane
Base acid sequence.The transmembrane peptides are the native sequences from mammalian cell, plant cell, yeast cells, or are manually set
Fusion protein can be fitted into the sequence of cytoplasma membrane by meter.
It is furthermore preferred that the transmembrane peptides are the Wbp1 cross-film peptide sequence from yeast cells, amino acid sequence such as SEQ
Shown in ID NO.2, specially are as follows:
TGEFILPDRHGVFTFLTDYRKIGLSFTTDKDVKAIRHLANDEYPRSWEISNSWVYISAICGVIVAWIF
FVVSFVTTSSVGKKLETFKKT。
Preferably, the effect protein structural domain intracellular refers to the protein structure domain of generation function after any complementation, is produced
Raw effect includes generating fluorescent ability, release transcription factor transcriptional reporter gene and generation chemiluminescence function.
It is furthermore preferred that the effect protein structural domain intracellular be fluorescin C-terminal and N-terminal structural domain, ubiquitin protein C-terminal with
N-terminal structural domain or luciferase N-terminal and C-terminal structural domain.
Preferably, the fluorescin is green fluorescent protein EGFP, yellow fluorescence protein YFP.
The amino acid sequence of the C-terminal domain C YFP of the yellow fluorescence protein YFP is as shown in SEQ ID NO.3, specifically
Are as follows: Dgsvqladhy qqntpigdgp vllpdnhyls yqsalskdpn ekrdhmvlle fvtaagitlg mdelyk.
The amino acid sequence of the N-terminal structural domain NYFP of the yellow fluorescence protein YFP is as shown in SEQ ID NO.4, specifically
Are as follows: Vskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt Gklpvpwptl
vttfgygvqc farypdhmkq hdffksampe gyvqertiff Kddgnyktra evkfegdtlv nrielkgidf
kedgnilghk leynynshnv Yimadkqkng ikvnfkirhn iedg。
Preferably, in step (1), the bait expression unit sequence contains the secretion signal being arranged successively from N-terminal to C-terminal
DNA encoding peptide sequence, bait coding gene sequence, transmembrane peptides coding gene sequence and effect protein structural domain intracellular coding
Gene order (as shown in Figure 2 A).Wherein, secreting signal peptide expressed by the bait expression unit is responsible for expressing bait simultaneously
Outside secretion to cell membrane, transmembrane peptides are responsible for connecting extracellular bait and effect protein structural domain (as shown in Figure 2 B) intracellular.Contain
The bait expression vector of the bait expressed sequence is suitble to the expression of natural endowment bottom secretory or soluble protein intracellular.
In the bait expression vector enumerated in the embodiment of the present invention, the bait expression unit sequence contains from N-terminal to C
Hold be arranged successively secreting signal peptide coding gene sequence, bait protein coding gene sequence, transmembrane peptides coding gene sequence, with
And effect protein structural domain coding gene sequence intracellular.The secreting signal peptide is the Wbp1 signal peptide sequence from yeast cells
Column, amino acid sequence are
MARVMRTDWNFFFCILLQAIFVVGTQTSRTLVLYSK (shown in SEQ ID NO.1).The cross-film peptide sequence
For the Wbp1 cross-film peptide sequence from yeast cells, amino acid sequence are as follows:
TGEFILPDRHGVFTFLTDYRKIGLSFTTDKDVKAIRHLANDEYPRSWEISNSWVYISAICGVIVAWIF
FVVSFVTTSSVGKKLETFKKT (shown in SEQ ID NO.2).The effect protein intracellular is fluorescin structural domain.
Preferably, the bait expression unit sequence contains the effect protein structural domain intracellular being arranged successively from N-terminal to C-terminal
Coding gene sequence, transmembrane peptides coding gene sequence, bait protein coding gene sequence.(as shown in Figure 2 C).Contain the fishing
The expression that the bait expression vector of bait expressed sequence is suitble under natural endowment for secretory or soluble protein intracellular.
Above two bait expression vector can express bait to extracellular, but the N of bait expressed by the two is last
End is towards on the contrary.
Preferably, the bait expression vector is that the bait expression unit is cloned into the yeast with that can screen label
On expression plasmid, building is obtained.
Specifically, bait expression vector of the present invention the preparation method comprises the following steps: by the method for PCR the two of bait expression unit
The insertion of bait expression unit is had coding DNA binding function domain by end addition restriction enzyme site sequence, the method using digestion, connection
The yeast two-hybrid plasmid of gene screens, identifies that the correct connection product of connection is bait expression vector of the invention.The fishing
Bait expression vector has the expression plasmid of yeast that can screen label.
Preferably, in step (2), the prey expression unit sequence contains the secretion signal being arranged successively from N-terminal to C-terminal
DNA encoding peptide sequence, prey coding gene sequence, transmembrane peptides coding gene sequence and effect protein structural domain intracellular coding
Gene order.Prey expression vector containing the prey expression unit sequence is for expressing non-memebrane protein.
Preferably, the prey expression unit sequence contains the effect protein structural domain intracellular being arranged successively from N-terminal to C-terminal
Coding gene sequence, transmembrane peptides coding gene sequence, prey coding gene sequence.Contain the prey expression unit sequence
Prey expression vector for expressing non-memebrane protein.
The N-terminal of prey expressed by above two prey expression vector is towards on the contrary.But two kinds of prey expression vectors
The albumen memebrane protein of those N, C-terminal simultaneously on the outside of cytoplasma membrane can be accurately expressed on cell membrane.
Preferably, the prey expression unit sequence contains the effect protein structural domain intracellular being arranged successively from N-terminal to C-terminal
Coding gene sequence, prey coding gene sequence.The N-terminal of prey expressed by the prey expression unit is intracellular.(such as Fig. 3 A
It is shown).Prey expression vector containing the prey expression unit sequence is for expressing embrane-associated protein.The prey expression carries
The albumen of those N, C-terminal simultaneously on the inside of cytoplasma membrane or in endoplasmic can be accurately expressed on cell membrane by body.
In the prey expression vector enumerated in a preferred embodiment of the present invention, effect protein structural domain intracellular is fluorescence
Protein structure domain.Specially yellow fluorescence protein structural domain.
Preferably, the prey expression unit sequence contains the effect protein structural domain intracellular being arranged successively from N-terminal to C-terminal
Coding gene sequence, transmembrane peptides coding gene sequence, prey coding gene sequence.Prey expressed by the prey expression unit
N-terminal extracellular.One section of transmembrane peptides is merged, enables to effect protein structural domain intracellular to be retained in intracellular.(as shown in Figure 3B).
Prey expression vector containing the prey expression unit sequence is for expressing embrane-associated protein.
In the prey expression vector enumerated in a preferred embodiment of the present invention, effect protein structural domain intracellular is fluorescence
Protein structure domain.Specially yellow fluorescence protein structural domain.
Preferably, the prey is embrane-associated protein, and the prey expression unit sequence contains successively arranges from N-terminal to C-terminal
The prey coding gene sequence of column, effect protein structural domain coding gene sequence intracellular.Expressed by the prey expression unit
The C-terminal of prey is intracellular.(as shown in Figure 3 C).
In the prey expression vector enumerated in a preferred embodiment of the present invention, effect protein intracellular is fluorescin.
Specially green fluorescent protein or yellow fluorescence protein.
Preferably, the prey is embrane-associated protein, and the prey expression unit sequence contains successively arranges from N-terminal to C-terminal
Prey protein coding gene sequence, the transmembrane peptides coding gene sequence of column, effect protein N-terminal structural domain encoding gene sequence intracellular
Column.The C-terminal of prey expressed by the prey expression vector is extracellular.One section of transmembrane peptides is merged, effect egg intracellular is enabled to
White N-terminal structural domain is retained in intracellular.(as shown in Figure 3D).
In the prey expression vector enumerated in a preferred embodiment of the present invention, effect protein structural domain intracellular is green
Fluorescin or yellow fluorescence protein.
Preferably, the prey expression unit is cloned into the yeast with coding selection markers by the prey expression vector
On expression plasmid, building is obtained.
Specifically, prey expression vector the preparation method comprises the following steps: being added by the method for PCR at the both ends of prey expression unit
The insertion of prey expression unit is had the expression plasmid of yeast for screening label by restriction enzyme site sequence, the method using digestion, connection,
It screens, identify that the correct connection product of connection is prey expression vector of the invention.
It should be noted that bait and prey are opposite address, the two is not different in itself.
Preferably, the bait or prey are cell factor, virus capsid protein or the something lost that can be expressed as above-mentioned substance
Pass substance.
Preferably, in some embodiments, signified cell membrane includes the cytoplasma membrane of eukaryon, protokaryon active somatic cell,
Or the cytoplasma membrane ingredient extracted after clasmatosis.
The technical system and realization step of memebrane protein yeast two-hybrid system are relatively complicated, this is mainly reflected in bait
The expression needs of albumen are accurately positioned on cell.That is the effect protein structural domain merged with bait protein must be positioned at
In cell membrane, downstream reaction could be started, and then realize that effect protein intracellular is converted into the biological effect of macroscopic view.
Existing memebrane protein yeast two-hybrid system can be used to study two memebrane proteins interaction and a film egg
The interaction of Bai Yuyi cytoplasmic protein.Any memebrane protein all can serve as bait, can make to merge with testing protein
Interaction module is located in cytoplasm.The effect protein structural domain end for the protein amalgamation and expression that existing system is studied must
It must be positioned in cytoplasm, the expression of reporter gene could be activated.Therefore, and not all memebrane protein is all suitable for the system, such as
Those N, C-terminal simultaneously protein on the outside of cytoplasma membrane or in endoplasmic then it is uncomfortable in this way.
However, as previously mentioned, then those N, C-terminal can be existed simultaneously using yeast two-hybrid system outside film of the invention
Albumen on the outside of cytoplasma membrane or in endoplasmic is accurately expressed on cell membrane.To be present in extracellular ligand sieve
Select receptor antagonist.
Second aspect of the present invention discloses method above-mentioned for the application in ligand screening receptor antagonist.
Preferably, the ligand is present in extracellularly.
Compared with prior art, the invention has the following beneficial effects:
(1) receptor and ligandin that this method is studied are to avoid in conventional method in body (in vivo) state
The loss of activity of ligandin caused by protein purification and markers step；
(2) the detected signal of this method is ligand and the direct interaction signal of receptor, avoids and examines in existing certain methods
The indirect signal that multiple signal transduction generates is surveyed, data reliability is improved；
(3) after being successfully established system, culture can be expanded by simple host strain and obtains a large amount of thallus, it is easy to established
Using 96 orifice plates or 384 orifice plate fluorescence microplate readers as the high flux screening platform of detector；
(4) this method is not required to consideration host cell membrane to the passability of antagonist, therefore is suitble to all kinds of test-compounds.
(5) present invention can be to be present in extracellular ligand screening receptor antagonist, and the receptor can be solubility
Receptor can also be transmembrane receptor.
In short, this method is a kind of receptor antagonist detection method for integrating stability, universality, high throughput；
Detailed description of the invention
Fig. 1: the antagonism schematic diagram of receptor and ligand interaction and antagonist: the screening of (A) membrane receptor antagonist is shown
It is intended to；(B) soluble recepter antagonist screens schematic diagram.
Fig. 2: bait protein-transmembrane peptides-effect protein expression pattern figure
Fig. 3: vector construction ideograph of the memebrane protein as prey protein
Fig. 4: bait protein and prey protein interaction schematic diagram
The vector construction and expression and localization of Fig. 5: CXCL12 ligandin
Fig. 6: CXCR4 receptor protein vector construction and expression and localization
Fig. 7: Chemokine CXCL12-and receptor CXCR 4 interaction system are established.(A) CXCL12-CYFP vector construction is illustrated
Figure.CXCL12 mature polypeptide coding sequence and secreting signal peptide, transmembrane signal peptide, CYFP transcription factor fusion constitute CXCL12-
CYFP expression vector；(B) CXCR4-NYFP vector construction schematic diagram.Acceptor gene (7 transmembrane receptors) C-terminal merges NYFP,
Constitute CXCR4-NYFP expression vector.(C) interact schematic diagram.CXCL12 and CXCR4 simultaneously expression and localization in cell membrane,
Interaction guides effect protein CYPF and NYFP intracellular complementary, induces bimolecular fluorescence complementary (BiFC) effect；(D)CXCL12
With OST1 without interaction schematic diagram.CXCL12 and OST1 gene are positioned at cell membrane surface simultaneously, but because CXCL12 with
There is no interactions by OST1, therefore not can induce BiFC effect.(E-H) laser co-focusing result.CXCL12-CXCR4 cell
Surface illustrates that interaction (E) has occurred in CXCL12 and CXCR4 there are yellow fluorescence；And there is no mutual by CXCL12 and OST1
Effect, therefore express bacterial strain surface and yellow fluorescence (F) is not present.Individually transfection CXCL12-CYFP carrier (G) or CXCR4-NYFP
Carrier (H), phage surface is without yellow fluorescence.(I) fluorescent strength determining.With spectrophotometric determination difference conversion bacterial strain
Yellow fluorescence intensity results.
Fig. 8: compound antagonism CXCR4 and CXCL12 Reaction kinetics research.(A) AMD3100 antagonism Time-activity-curve figure.No
AMD3100 and CXC12-CXCR4 cell strain with concentration is incubated for, with the performance of incubation time, CXCL12-CXCR4 compound
Concentration gradually decreases, and balance is reached when reaction is carried out to 25min.(B) amount effect curve of difference CXCL12-CXCR4 antagonist.
When equilibrium is reached, CXCL12-CXCR4 complex concentration (fluorescence intensity) is increased with Antagonist concentration and is dropped in reaction system
Low, curve is in " anti-S " curve.The 50 3nhibitory dose IC50 of each compound of median effective dose, knot are calculated by software
Fruit is consistent with having been reported.
Specific embodiment
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Technical solution of the present invention is further described below by specific embodiment.
The vector construction and expression and localization of embodiment 1.CXCL12 ligandin
Purpose: by reaching CXCL12 gene and signal peptide gene, transmembrane peptides gene fusion expression and being positioned at cell membrane
Effect.
Plasmid construction: by mouse chemokine CXCL12 mature polypeptide coding sequence (shown in SEQ ID NO.5, specifically:
KPVSLSYRC PCRFFESHIA RANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ EYLEKALNK) expand
Increase and be sequenced, with secretion signal peptide sequence (come from yeast Wbp1 signal peptide sequence, shown in SEQ ID NO.1, specifically:
MARVMRTDWNFFFCILLQAIFVVGTQTSRTLVLYSK) transmembrane peptides (come from yeast Wbp1 cross-film peptide sequence,
Shown in SEQ ID NO.2, specifically:
TGEFILPDRHGVFTFLTDYRKIGLSFTTDKDVKAIRHLANDEYPRSWEISNSWVYISAICGVIVAWIF
FVVSFVTTSSVGKKLETFKKT it) merges, connects green fluorescent protein (EGFP) reporter gene, entire fusion protein gram thereafter
It is grand to form structure as shown in Figure 5A into Yeast expression carrier pGAD-T7, it is denoted as CXCL12-EGFP plasmid.
The prediction of fusion protein transmembrane structure: the cross-film topology knot of TMHMM2.0 on-line analysis software analysis fusioning protein is used
Structure.Software calculated result shows that the fusion protein can be reached and expresses CXCL12 protein part to cell membrane, and passes through
The topological structure (Fig. 5 B) that transmembrane peptides are connect with EGFP on the inside of cell membrane.
Plasmid transfection: by LiAc method for transformation convert CXCL12-EGFP plasmid to GoldY2H cell strain (MAT α,
Trp1-901,Leu2-3,112,ura3-52,his3-200,gal4Δ,gal80Δ,LYS2::GAL1UAS-Gal2TATA-
His3,GAL2UAS-Gal2TATA-Ade2,URA3::MEL1USA-Mel1TATA, AUR1-C, MEL1), transformant is in SD Leu-Nutrition
It is screened 5 days for 30 degrees Celsius on deficiency culture medium.
Cellular localization observation: the cellular localization of fluorescence microscope green fluorescent protein.As shown in Figure 5 C, result is observed
It has been shown that, cell membrane surface have apparent green florescent signal, it was demonstrated that the correct expression that CXCL12 bait protein can be as would be expected
In cell membrane surface.
Embodiment 2.CXCR4 receptor protein vector construction and positioning
Purpose: expression and localization of the clear CXCR4 receptor in yeast cells.
Vector construction: CXCR4 acceptor gene is cloned, and is merged with yellow fluorescence protein (YFP), and Yeast expression is cloned into
In carrier pGBK-T7 (Fig. 6 A), it is denoted as: CXCR4-YFP plasmid.Wherein, the relevant information of CXCR4 is detailed in:
CXCR4 referenceHTTP://WWW.UNIPROT.ORG/UNIPROT/P70658；(NP_034041.2)
The prediction of fusion protein transmembrane structure: the cross-film topology knot of TMHMM2.0 on-line analysis software analysis fusioning protein is used
Structure.Software calculated result shows that the fusion protein can correctly express receptor protein to cell membrane, and C-terminal is merged
YFP protein expression is to (Fig. 6 B) intracellular.
Plasmid transfection: CXCR4-YFP plasmid is converted to Y187 cell strain by LiAc method for transformation, transformant is in SD-
Trp-It is screened 5 days for 30 degrees Celsius on auxotroph culture medium.Monoclonal bacterial strain is selected for Fluirescence observation.
Cellular localization observation: using laser confocal microscope, with the cellular localization of 514nm exciting light observation YFP.Observation
The results show that strain cell film surface has apparent yellow fluorescence signal, it was demonstrated that CXCR4 albumen being capable of correct table as prospectively
Up in cell membrane surface.
Embodiment 3.CXCL12 and CXCR4 interaction detection platform is established
Embodiment purpose: CXCL12 and CXCR4 interaction detection platform is established by bimolecular fluorescence complementary technology, is used
In subsequent receptor antagonist screening.
Principle: ligand gene CXCL12 (NP_038683.1) passes through transmembrane peptides and yellow fluorescence protein C-terminal structural domain
(CYFP) it merges, receptor CXCR 4 (NP_034041.2) is merged with yellow fluorescence protein N-terminal structural domain (NYFP).NYFP and CYFP
It individually cannot fluoresce, only when CXCL12 and CXCR4 interact, NYFP and CYFP generation bimolecular fluorescence are mutual
It mends (BiFC), rebuilding becomes the complete active yellow fluorescence protein (YFP) with fluorescent functional.It is detected by fluorescence microplate reader
Bacterium solution yellow fluorescence (529 nanometers of wavelength) intensity.
Plasmid construction: CXCL12-CYFP plasmid construction:
By mouse chemokine CXCL12 mature polypeptide coding sequence (shown in SEQ ID NO.5, specifically: KPVSLSYRC
PCRFFESHIARANVKHLKIL NTPNCALQIV ARLKNNNRQVCIDPKLKWIQ EYLEKALNK) N-terminal and secretion believe
Number peptide sequence (yeast Wbp1 signal peptide sequence is come from, shown in SEQ ID NO.1, specifically: MARVMRTDWNFFFCILLQAIF
VVGTQTSRTLVLYSK), C-terminal and transmembrane signal peptide sequence (come from yeast Wbp1 cross-film peptide sequence, shown in SEQ ID NO.2,
Specifically:
TGEFILPDRHGVFTFLTDYRKIGLSFTTDKDVKAIRHLANDEYPRSWEISNSWVYISAICGVIVAWIF
FVVSFVTTSSVGKKLETFKKT it) merges, merges yellow fluorescence protein C-terminal structural domain (CYFP) thereafter, entire fusion protein base
Because being cloned into Yeast expression carrier PGAD-T7, formed such as figure: structure shown in 7A is denoted as CXCL12-CYFP plasmid.
CXCR4-NYFP plasmid construction: yellow fluorescence protein N-terminal knot is merged in mouse chemokine receptor CXCR 4 N-terminal
Structure domain NYFP, fusion gene cloning name CXCR4-NYFP into Yeast expression carrier pGBK-T7.
The protein structure that it is expressed is as shown in Figure 7 B.
Control vector building:
Negative control:
Using unrelated yeast memebrane protein OST1 (HTTP://WWW.UNIPROT.ORG/UNIPROT/P41543)(NP_
012532.3) fusion NYFP constitutes negative control vector, it is expected that expression structure is as shown in Figure 7 D.Because of OST and CXCL12
Theoretically without interaction.
Plasmid transfection and positive colony screening: pass through LiAc transfection method cotransfection CXCL12-CYFP and CXCR4-NYFP
Or OST1-NYFP carrier, to Y187 yeast cells strain, thallus is in double defect culture medium SD Leu-Trp-Upper 30 degrees Celsius of cultures 5d,
Picking monoclonal colonies identify plasmid co-transfection by PCR.CXCL12-CYFP or CXCR4-NYFP are individually transfected to Y187
Bacterial strain, as control.
The detection of bimolecular fluorescence complementary: picking monoclonal colonies use confocal laser scanning microscope cell membrane yellow
Fluorescence detects bacterium solution yellow fluorescence intensity using fluorescence microplate reader.
As a result:
There is yellow fluorescence in CXCL12-CYFP and CXCR4-NYFP coexpression strain cell film surface, CXCL12-CYFP with
OST1-NYFP coexpression bacterial strain does not have yellow fluorescence, and the bacterial strain for individually transfecting CXCL12-CYFP or CXCR4-NYFP is not observed
To cell membrane surface fluorescence.
Yellow fluorescence intensity in the bacterium solution of identical cell density is measured using fluorescence microplate reader, it is again seen that CXCL12-
CYFP and CXCR4-NYFP coexpression bacterial strain has high-intensitive yellow fluorescence signal compared with the control, and single plasmid transfects bacterium
Strain is compared with empty bacterium strain without apparent fluorescence signal.
Illustrate that CXCL12 has mediated CYFP and NYFP complementary with CXCR4 interaction, generates yellow fluorescence.
50 3nhibitory dose (IC50) measurement that 4. antagonist of embodiment combines CXCL12-CXCR4
Embodiment purpose: CXCR4 antagonist AMD3100, AMD070 and AMD3465 is to CXCL12 and CXCR4 interaction for measurement
The IC50 of inhibiting effect.
Material prepares: by 3 kinds of CXCR4 antagonists (AMD3100, AMD070 and AMD3465) and control compound NBI-
74330, the structural formula of four compounds is as shown below:
Various concentration gradient is dissolved to PBS buffer solution；The CXCL12-CXCR4 interaction system that embodiment 3 is established
Host strain of uniting expands culture, and thalline were collected by centrifugation, is resuspended with PBS buffer solution, prepares outstanding as the bacterium of absorbance OD600=0.5
Liquid.
Interaction antagonism: it by the test-compound solution 100ul of various concentration, mixes, adds to isometric bacterium solution
96 orifice plates.37 degrees Celsius of shakings, different time points sample detection on shaking table.Each concentration carries out 3 parallel hole experiments.
Binding kinetics research: fluorescence microplate reader measurement cell yellow fluorescence intensity (excitation wavelength 514nm, hair are used
Third contact of a total solar or lunar eclipse wavelength 525nm).First using the reaction time as abscissa, maps by ordinate of fluorescence intensity, determine that reaction process reaches
Time needed for balance.Secondly it using compound concentration as abscissa, maps, obtains using fluorescence intensity after reaction balance as ordinate
Amount effect curve.The IC50 of compound antagonism CXCL12 and CXCR4 interaction is calculated using biological software.
As a result:
The results show that fluorescent value is gradually reduced at any time, In after various concentration AMD3100 mixes incubation with bacteria suspension
Reach plateau (Fig. 8 A) at 25 minutes.Illustrate competitively to combine with CXCR4, receptor is matched after antagonist compound is added
Body, antagonist three association and dissociation balance is reached after 25min.
Using Test compound concentrations as abscissa, (Fig. 8 B) is mapped by ordinate of relative intensity of fluorescence.It can be with from curve
Find out, three kinds of test-compounds all have antagonism to the interaction of CXCL12 and CXCR4, with the increasing of drug concentration
Add, yellow fluorescence intensity is gradually reduced.And control compound is without apparent inhibiting effect.Three kinds of inhibitor are calculated by software
IC50, result are that AMD3100 is 34.5 ± 2.4nM；AMD3465 is 123.8 ± 5.3nM；AMD070 is 14.1 ± 1.9nM.It should
As a result it matches (referring to Fricker, S.P.et al.Biochem Pharmacol.2006 Aug 28 with having been reported；72
(5):588-96；Rosenkilde MM,et al.J Biol Chem.2007.282(37):27354-65；Skerlj,R.et
al.Bioorg Med Chem Lett 2011.21(1):262-6)。
This result shows that this system can be used in study compound the CXCL12-CXCR4 antagonism to interact is ground
Study carefully, can get Antagonistic reaction equilibration time, two important parameters of IC50 of antagonist.
Claims (5)
1. a kind of method to be present in extracellular ligand screening receptor antagonist, the method specifically comprise the following steps:
(1) using ligand as bait, construct bait expression vector: the construction method is selected from following any: A. will secretion letter
Number DNA encoding peptide sequence, transmembrane peptides coding gene sequence and bait coding gene sequence and effect protein structural domain intracellular are compiled
Bait expression unit sequence is obtained after code gene order fusion, and the bait expression unit is cloned into have and encodes screening mark
On the expression plasmid of yeast of label, building obtains the bait expression vector of expression bait, and the bait expression unit sequence contains from N
The secreting signal peptide coding gene sequence of holding C-terminal to be arranged successively, bait coding gene sequence, transmembrane peptides coding gene sequence,
And effect protein structural domain coding gene sequence intracellular；B. by transmembrane peptides coding gene sequence and bait coding gene sequence with
And bait expression unit sequence is obtained after effect protein structural domain coding gene sequence fusion intracellular, and the bait is expressed into list
Member is cloned on the expression plasmid of yeast with coding screening label, and building obtains the bait expression vector of expression bait, described
Bait expression unit sequence contains effect protein structural domain coding gene sequence intracellular, the transmembrane peptides being arranged successively from N-terminal to C-terminal
Coding gene sequence, bait protein coding gene sequence；
(2) it using receptor as prey, constructs prey expression vector: secreting signal peptide coding gene sequence, transmembrane peptides is encoded into base
Because at least one of sequence is merged and is obtained with prey coding gene sequence and effect protein structural domain coding gene sequence intracellular
It obtains prey expression unit or the prey expression unit sequence is by prey coding gene sequence and effect protein knot intracellular
Structure domain coding gene sequence obtains prey expression unit sequence after directly merging, and the prey expression unit is cloned into has
On the expression plasmid of yeast of coding screening label, building obtains the prey expression vector of expression prey, the prey expression unit
Sequence further includes any one of following characteristics: a) when prey expression vector is for when expressing non-memebrane protein, the prey to be expressed
Unit sequence contains the secreting signal peptide coding gene sequence being arranged successively from N-terminal to C-terminal, prey coding gene sequence, cross-film
DNA encoding peptide sequence and effect protein structural domain coding gene sequence intracellular；B) non-for expressing when prey expression vector
When memebrane protein, the prey expression unit sequence contains the effect protein structural domain intracellular coding base being arranged successively from N-terminal to C-terminal
Because of sequence, transmembrane peptides coding gene sequence, prey coding gene sequence；C) when prey expression vector is for expressing embrane-associated protein
When, the prey expression unit sequence contains the effect protein structural domain encoding gene sequence intracellular being arranged successively from N-terminal to C-terminal
Column, prey coding gene sequence；D) when prey expression vector is used to express embrane-associated protein, the prey expression unit sequence
Containing the effect protein structural domain coding gene sequence intracellular being arranged successively from N-terminal to C-terminal, transmembrane peptides coding gene sequence, hunt
Object coding gene sequence；E) when prey expression vector is for when expressing embrane-associated protein, the prey expression unit sequence to contain
Prey coding gene sequence, the effect protein structural domain coding gene sequence intracellular being arranged successively from N-terminal to C-terminal；F) work as prey
Expression vector is for when expressing embrane-associated protein, the prey expression unit sequence to contain the prey being arranged successively from N-terminal to C-terminal
Protein coding gene sequence, transmembrane peptides coding gene sequence, effect protein N-terminal structural domain coding gene sequence intracellular；
(3) by gained prey expression vector in gained bait expression vector in step (1) and step (2) be transferred in yeast strain into
Interaction occurs outside cell membrane for row coexpression, expressed ligand and receptor, and it is complementary to draw effect protein intracellular；
(4) detect antagonist activities as measurement complementary effect: bait expression obtained by obtained stable expression step (1) carries
Antagonist candidate is added in the positive yeast of gained prey expression vector in body and step (2), calculates the half suppression of candidate
Amount IC50 processed, thus it is speculated that the affinity Ki or dissociation COEFFICIENT K D of candidate and ligand filter out the receptor antagonist of ligand.
2. the method as described in claim 1, which is characterized in that further include any one of following characteristics or multinomial: 1) described
Effect protein structural domain intracellular is fluorescin C-terminal and N-terminal structural domain, ubiquitin protein C-terminal and N-terminal structural domain or luciferase N
End and C-terminal structural domain；2) secreting signal peptide is the Wbp1 signal peptide sequence from yeast cells, and amino acid sequence is such as
Shown in SEQ ID NO.1；3) transmembrane peptides are the Wbp1 cross-film peptide sequence from yeast cells, amino acid sequence such as SEQ
Shown in ID NO.2.
3. the method as described in claim 1, which is characterized in that the yeast strain is Y2HGold or Y187.
4. method as described in claim 1-3 any claim is for the application in ligand screening receptor antagonist.
5. application as claimed in claim 4, the ligand are present in extracellularly.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1221449A (en) * | 1996-04-02 | 1999-06-30 | 依达研究发展有限公司 | Modulators of TNF receptor associated factor (TRAF), their preparation and use |
| CN1384878A (en) * | 1999-09-02 | 2002-12-11 | 耶达研究发展有限公司 | IREN protein, its preparation and use |
| WO2004092394A3 (en) * | 2003-04-08 | 2005-03-31 | Philadelphia Children Hospital | Fluid planar lipid layer-based membrane-anchored ligand system with specific binding pair members and methods of use thereof |
| US7169567B1 (en) * | 1998-12-31 | 2007-01-30 | The General Hospital Corporation | Screening assay utilizing the PTH receptor |
| US7700728B2 (en) * | 2005-03-24 | 2010-04-20 | Schering Corporation | Use of chimeric receptors in a screening assay for identifying agonists and antagonists of cell receptors |
| CN102112490A (en) * | 2008-07-08 | 2011-06-29 | 昂考梅德药品有限公司 | Notch1 receptor binding agents and methods of use thereof |
| CN103290034A (en) * | 2012-03-01 | 2013-09-11 | 华中农业大学 | Ubiquitin ligase gene participating in symbiotic nodulation of leguminous plants, and application thereof |
-
2015
- 2015-02-12 CN CN201510075993.3A patent/CN105986001B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1221449A (en) * | 1996-04-02 | 1999-06-30 | 依达研究发展有限公司 | Modulators of TNF receptor associated factor (TRAF), their preparation and use |
| US7169567B1 (en) * | 1998-12-31 | 2007-01-30 | The General Hospital Corporation | Screening assay utilizing the PTH receptor |
| CN1384878A (en) * | 1999-09-02 | 2002-12-11 | 耶达研究发展有限公司 | IREN protein, its preparation and use |
| WO2004092394A3 (en) * | 2003-04-08 | 2005-03-31 | Philadelphia Children Hospital | Fluid planar lipid layer-based membrane-anchored ligand system with specific binding pair members and methods of use thereof |
| US7700728B2 (en) * | 2005-03-24 | 2010-04-20 | Schering Corporation | Use of chimeric receptors in a screening assay for identifying agonists and antagonists of cell receptors |
| CN102112490A (en) * | 2008-07-08 | 2011-06-29 | 昂考梅德药品有限公司 | Notch1 receptor binding agents and methods of use thereof |
| CN103290034A (en) * | 2012-03-01 | 2013-09-11 | 华中农业大学 | Ubiquitin ligase gene participating in symbiotic nodulation of leguminous plants, and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo;IGOR STAGLJAR 等;《Proc. Natl. Acad. Sci. USA》;19980430;第95卷;第5187-5192页 * |
| MEK2与猪癟病毒E2蛋白的相互作用及其对猪痕病毒ft制的调控作用;廖亚金;《中国畜牧兽医学会家畜传染学分会第八届全国会员代表大会暨第十五次学术研讨会论文集》;20131231;正文第5-7、11-15页 * |
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